Les Henderson

Randomized Phase Ib/II Study of Gemcitabine Plus Placebo or Vismodegib, a Hedgehog Pathway Inhibitor, in Patients With Metastatic Pancreatic Cancer

Purpose Sonic hedgehog (SHH), an activating ligand of smoothened (SMO), is overexpressed in > 70% of pancreatic cancers (PCs). We investigated the impact of vismodegib, an SHH antagonist, plus gemcitabine (GV) or gemcitabine plus placebo (GP) in a multicenter phase Ib/randomized phase II trial and preclinical PC models. Patients and Methods Patients with PC not amenable to curative therapy who had received no prior therapy for metastatic disease and had Karnofsky performance score ≥ 80 were enrolled. Patients were randomly assigned in a one-to-one ratio to GV or GP. The primary end point was progression-free-survival (PFS). Exploratory correlative studies included serial SHH serum levels and contrast perfusion computed tomography imaging. To further investigate putative biologic mechanisms of SMO inhibition, two autochthonous pancreatic cancer models (KrasG12D; p16/p19fl/fl; Pdx1-Cre and KrasG12D; p53R270H/wt; Pdx1-Cre) were studied. Results No safety issues were identified in the phase Ib portion (n = 7), and the phase II study enrolled 106 evaluable patients (n = 53 in each arm). Median PFS was 4.0 and 2.5 months for GV and GP arms, respectively (95% CI, 2.5 to 5.3 and 1.9 to 3.8, respectively; adjusted hazard ratio, 0.81; 95% CI, 0.54 to 1.21; P = .30). Median overall survival (OS) was 6.9 and 6.1 months for GV and GP arms, respectively (95% CI, 5.8 to 8.0 and 5.0 to 8.0, respectively; adjusted hazard ratio, 1.04; 95% CI, 0.69 to 1.58; P = .84). Response rates were not significantly different. There were no significant associations between correlative markers and overall response rate, PFS, or OS. Preclinical trials revealed no significant differences with vismodegib in drug delivery, tumor growth rate, or OS in either model. Conclusion The addition of vismodegib to gemcitabine in an unselected cohort did not improve overall response rate, PFS, or OS in patients with metastatic PC. Our preclinical and clinical results revealed no statistically significant differences with respect to drug delivery or treatment efficacy using vismodegib.

Durable Complete Response of Metastatic Gastric Cancer with Anti-Met Therapy Followed by Resistance at Recurrence

UNLABELLED A 48 year-old female with chemo-refractory metastatic gastric cancer to the liver was treated on a Phase I clinical trial with MetMAb, a monoclonal antibody targeting the Met tyrosine kinase receptor. The primary tumor had high MET gene polysomy and evidence for an autocrine production of HGF, the growth factor ligand of Met. A complete response was obtained lasting two years; the cancer recurred as a peritoneal deposit invading into the transverse colon and a gastrohepatic ligament node. Compassionate use of MetMAb therapy at recurrence achieved a mixed response--a partial response of the two initial lesions, but with development of multiple new foci of carcinomatosis. Tissue and serum studies evaluating the Met signaling pathway did correlate with MetMAb treatment response initially and at the time of recurrence. SIGNIFICANCE This research brief is the first to describe a durable complete response obtained with a molecularly targeted monoclonal antibody, MetMAb, to the receptor tyrosine kinase, Met, in a patient with chemorefractory metastatic gastric cancer. It is also the first to report biomarkers that predicted therapeutic response to Met inhibition.

Copy number variation of CCL3-like genes affects rate of progression to simian-AIDS in Rhesus Macaques (Macaca mulatta).

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.

RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly over-expressed in 74% of gastroesophageal samples (n=94), and over-expression was prognostic of poor survival (p=0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number by quantitative polymerase chain reaction, and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 gene copy number. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors, and proved synergistic when combined with STAT3 inhibition (combination index <1). These preclinical studies define RON as an important novel prognostic marker and therapeutic target for gastroesophageal cancer warranting further investigation.

Absolute Quantitation of Met Using Mass Spectrometry for Clinical Application: Assay Precision, Stability, and Correlation with MET Gene Amplification in FFPE Tumor Tissue

Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification. Conclusions The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

Paxillin expression and amplification in early lung lesions of high-risk patients, lung adenocarcinoma and metastatic disease

Background Paxillin is a modular protein that localises to cell adhesion sites where it facilitates bidirectional communication between the intracellular actin cytoskeleton and the extracellular matrix. These complex and dynamic interactions are essential for cell adhesion, cell migration and cell survival. The authors have previously demonstrated that paxillin is overexpressed in lung cancer tissues and identified somatic paxillin mutations in 9% of lung cancers. A murine in vivo xenograft model of the most common paxillin mutation (A127T) showed increased cell proliferation and invasive tumour growth, establishing an important role for paxillin in the development of lung cancer. Methods The authors analysed 279 bronchoscopy-aided biopsy specimens from 92 high-risk patients. Adenocarcinoma with bronchioloalveolar features and pure bronchioloalveolar carcinoma (BAC) were analysed with fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC). Results Paxillin is overexpressed in premalignant areas of hyperplasia, squamous metaplasia and goblet cell metaplasia, as well as dysplastic lesions and carcinoma in high-risk patients. Concordance between increased paxillin gene copy number and paxillin overexpression was observed in cases of adenocarcinoma eusomic for chromosome 12. Conclusions Paxillin overexpression occurs during the earliest stages of lung cancer development. FISH and IHC analysis of lung adenocarcinoma suggests that relatively small-scale genomic rearrangements of chromosome 12 are associated with paxillin overexpression in lung adenocarcinoma.

Targeting wild-type KRAS -amplified gastroesophageal cancer through combined MEK and SHP2 inhibition

The role of KRAS, when activated through canonical mutations, has been well established in cancer1. Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas2–4. KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS–GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.Amplification of wild-type KRAS in a subset of gastroesophageal tumors drives intrinsic resistance to MAPK inhibition that can be overcome by combined targeting of MEK and SHP2.

Abstract 1207: Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues.

Aberrant over-expression of receptor tyrosine kinases, (e.g. MET, HER, FGFR, and IGFR) as well as other oncogenic mediators (e.g. KRAS, PI3 Kinase and SRC) are known drivers of gastroesophageal adenocarcinoma (GEC), subdividing the disease into distinct molecular subsets. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best. Moreover, IHC of multiple proteins of interest is laborious, time consuming, wasteful of scarce tissue, and costly. We present a quantitative mass spectrometric (MS) assay for FFPE GEC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a panel of gastroesophageal cancer (GEC) cell lines and tissues. Using trypsin digestion mapping of recombinant oncoproteins, we identified unique peptide sequences, and built quantitative MS assays which could be multiplexed into a single SRM analysis of 1μg of tumor protein. Assays were preclinically validated on 10 different formalin fixed (FF) cell lines. We then tested the GEC-plex assay on a panel of FFPE GEC cell lines characterized by immunoblot (IB), IHC, and gene copy number by FISH. In addition to RON, we multiplexed SRM quantification of Met, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC. We evaluated 17 GEC lines including AGS wild type, scrambled shRNA (AGS-SC) and RON shRNA knockdown (AGS-KD) to assess ‘post-treatment’ changes in oncogene expression. We then evaluated 100 GEC human cancer tissues with paired peritoneal metastases when available and select paraneoplastic normal tissues using laser microdissection of tumor tissue from a single unstained 10μm thick section. Validation of the GEC-plex SRM assay on GEC cell lines revealed very high concordance when compared to IB and IHC measurement. The AGS-WT/SC cells showed comparable levels of RON (284/323 amol/μg cell protein), while RON was not detected in AGS-KD cells, as expected. Measurement of oncoproteins in GEC cell lines and tissues correlated well with IHC and FISH data. Multiplex oncogene quantification of all cell lines and tissues, along with expression profile changes in the AGS RON KD line compared to AGS-WT/SC will be presented. Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FF cells. The GEC-plex multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue from clinical samples, and is a novel clinically applicable approach for tumor characterization for baseline and post-treatment assessment. Citation Format: Daniel V. Catenacci, Peng Xu, Les Henderson, Wei-Li Liao, Sheeno Thyparambil, Jon Burrows, Todd Hembrough. Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2013-1207

Author Correction: Targeting wild-type KRAS -amplified gastroesophageal cancer through combined MEK and SHP2 inhibition

In the Supplementary Information originally published with this article, a lane was missing in the β-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.

Her2 expression in gastroesophageal cancer (GEC) FFPE tissue using mass spectrometry (MS) and correlation with HER2 gene amplification.

82 Background: HER2+ GEC derived benefit from trastuzumab. Her2 IHC is semi-quantitative, subjective, and sensitive to antigen instability; HER2 FISH is laborious, expensive, and subjective. False positivity/negativity have been described. Also, these are low throughput assays; there is known molecular heterogeneity, with several putative biomarkers, and only scarce tissue to assess for each. We sought to evaluate the association of Her2 MS expression with HER2 FISH, along with other markers within the ‘GEC-plex’. Methods: We utilized a previously described unique Her2 peptide and quantification method (Hembrough et al J Clin Oncol 32,2014(suppl 3;abstr17)). The assay was run on 27 cell lines, in parallel with HER2:CEP17 FISH. Her2 expression thresholds were established for HER2 amplification using ROC curves. We adjusted for Her3, Egfr, and Met MS expression levels and sample HER2:CEP17 ratio heterogeneity in a multiple linear regression model. The model/cut-offs were then validated prospectively on GEC ...